The Establishment And Application Of A Cell Cycle Synchronization Model Using Human Endometrial Stromal Cells

Decidualization refers to the process during which fibroblastic endometrial stromal cells differentiate into round,secretory decidual cells.Decidualization plays a crucial role in embryo implantation and maintenance of pregnancy,impairment of this process causes failure of implantation or placentation,and further leads to pregnancy loss or even infertility.Cell cycle highly associates with the process of decidualization,during which changed expression of many cell cycle-related factors have been reported.Difference in population of cells in different cell cycle phases at the initiation of decidualization is a possible reason that causes the variable results in studies using in vitro decidualized human endometrial stromal cells.To investigate the difference of endometrial stromal cells at different phases of the cell cycle in response to in vitro decidualization,we have established a synchronized cell cycle model for the in vitro decidualization studies using the endometrial stromal cell line ATCC CRL-4003(4003 cells).Four different methods were used to synchronize the cells: serum starvation,lovastatin,dual thymidine blocking,and serum starvation plus thymidine treatment.Our results showed that although the serum starvation method is able to synchronize most of the cells to the G0/G1 phase,these cells were no longer well synchronized when moved to S and G2/M phases.This may because of the difference in time that needed by difference cells to recover from starving,supported by the morphological changes of cells after starvation;lovastatin treatment can synchronize cells to G0/G1 phase,but the efficient dose of lovastatin is highly toxic to the cells;Compare to lovastatin,the double thymidine blocking method has much lower cytotoxicity,but we were unable to find an appropriate length of culture time between two thymidine treatments,it seems the precondition of using this method which requires the period of S phase be shorter than a total of the rest three phases of cell cycle is not the case in 4003 cells.Then,we have created the serum starvation plus thymidine method by combining the advantages of two different methods,this is a new method that we find is able to synchronize cells to G0/G1 phase,and keep the cells well synchronized to S phase and G2/M phase after a certain period of culture in normal culture medium.However,there was still a small population of cells stayed in G0/G1 phase without moving into cell cycle progression,it is presumed to be a set of dormant cells in their G0 phases.Three groups of cells that were synchronized into G0/G1 phase,S phase or G2/M phase by starvation plus thymidine method were used for in vitro decidualization for 8 h,respectively,The results showed that cells in different cell cycle phases were found to have different expression in IGFBP1 and FOXO1.In this study,a new cell cycle synchronization model of 4003 cells was established by the united starvation and thymidine methods,and three groups of cells in G0/G1,S,or G2/M phases were obtained,respectively.Cells in different cell cycle phases were found to have different responses to decidualization.Our results contribute to the understanding of the changes in the cell cycle during decidualization and the effects of cell cycle on decidualization,laying a foundation for further exploring the relationship between cell cycle and decidualization.